Interaction between sensory C-fibers and cardiac mast cells in ischemia/reperfusion: activation of a local renin-angiotensin system culminating in severe arrhythmic dysfunction

Abstract

Renin, the rate-limiting enzyme in the activation of the renin-angiotensin system (RAS), is synthesized and stored in cardiac mast cells. In ischemia/reperfusion, cardiac sensory nerves release neuropeptides such as substance P that, by degranulating mast cells, might promote renin release, thus activating a local RAS and ultimately inducing cardiac dysfunction. We tested this hypothesis in whole hearts ex vivo, in cardiac nerve terminals in vitro, and in cultured mast cells. We found that substance P-containing nerves are juxtaposed to renin-containing cardiac mast cells. Chemical stimulation of these nerves elicited substance P release that was accompanied by renin release, with the latter being preventable by mast cell stabilization or blockade of substance P receptors. Substance P caused degranulation of mast cells in culture and elicited renin release, and both of these were prevented by substance P receptor blockade. Ischemia/reperfusion in ex vivo hearts caused the release of substance P, which was associated with an increase in renin and norepinephrine overflow and with sustained reperfusion arrhythmias; substance P receptor blockade prevented these changes. Substance P, norepinephrine, and renin were also released by acetaldehyde, a known product of ischemia/reperfusion, from cardiac synaptosomes and cultured mast cells, respectively. Collectively, our findings indicate that an important link exists in the heart between sensory nerves and renin-containing mast cells; substance P released from sensory nerves plays a significant role in the release of mast cell renin in ischemia/reperfusion and in the activation of a local cardiac RAS. This culminates in angiotensin production, norepinephrine release, and arrhythmic cardiac dysfunction.

Fig. 1.

Renin-containing mast cells are located near substance P-positive nerves in the rat heart. Immunofluorescence of a rat atrial section stained with avidin conjugated to fluorescein (green) to identify mast cells. A, anti-substance P conjugated to Alexa Fluor 594 (red). B, anti-renin conjugated to Alexa Fluor 594 (red). Note in A that the substance P neuronal pattern is shown by the white arrow. Nuclei were counterstained with 4,6-diamidino-2-phenylindole. Scale bar = 10 μm.

Fig. 2.

Chemical stimulation of sensory C-fibers with capsaicin elicits the release of CGRP (A) and substance P (B) in ex vivo guinea pig hearts. Bars (means ± S.E.M.; n = 6) represent overflows of CGRP and substance P into the coronary effluent. Capsaicin (100 nM) was perfused for 6 min, and the coronary effluent was collected at 2-min intervals. Control bars (means ± S.E.M.; n = 6) represent the basal release of CGRP and substance P before perfusion with capsaicin. *, P < 0.05, **, P < 0.01, from control by one-way ANOVA followed by Dunnett's post-test analysis.

Fig. 3.

Chemical stimulation of sensory C-fibers with capsaicin elicits release of mast cell renin (A), ANG II formation (A and B), and NE release (A) in ex vivo guinea pig hearts. Bars (means ± S.E.M.) represent overflows of renin (angiotensin I formed) and NE into the coronary effluent. Capsaicin (100 nM) was perfused for 6 min alone (n = 8) or in the presence of the mast cell stabilizer cromolyn (300 μM, n = 5), the NK₁-receptor antagonist CP99994 [Anti-substance P (anti-SP); 100 nM, n = 5], the CGRP-receptor antagonist CGRP_8–37 (Anti-CGRP; 100 nM, n = 3), the H₃-receptor agonist imetit (100 nM, n = 6), or the ANG II AT₁-receptor antagonist EXP3174 (EXP; 10 nM, n = 4). Control bars (means ± S.E.M.; n = 31). Basal release of NE was 0.69 pmol/g. *, P < 0.05, **, P < 0.01, from capsaicin alone by one-way ANOVA followed by Dunnett's post-test analysis.

Fig. 4.

Substance P (SP) causes degranulation and renin release in HMC-1 by activating NK₁ receptors. Incubation of HMC-1 cells with substance P for 20 min elicits the release of renin and β-hexosaminidase (an index of mast cell degranulation) in a concentration-dependent manner. Points represent the response to substance P in the absence (closed squares) and presence (open circles) of the NK₁-receptor antagonist CP99994 (100 nM). A, points are means (±S.E.M.; n = 8–11) of percentage increases in renin release above basal level (basal renin activity, 1640 pg/h/mg protein ± 295; n = 26). B, points are means (± S.E.M.; n = 4–13) of percentage increases in β-hexosaminidase release above basal level (4.89 ± 0.41; n = 16). *, P < 0.05, **, P < 0.01, from basal by one-way ANOVA followed by Dunnett's post-test analysis. #, P < 0.05, ###, P < 0.001 from substance P by one-way ANOVA followed by Bonferroni's post-test analysis.

Fig. 5.

Ischemia/reperfusion causes the release of CGRP and substance P from ex vivo guinea pig hearts, and this is unaffected by the blockade of CGRP and NK₁ receptors. Bars (means ± S.E.M.) represent overflows of CGRP and substance P into the coronary effluent of isolated guinea pig hearts. Hearts were subjected to ischemia/reperfusion [i.e., 20-min global ischemia (Isch) followed by 30-min reperfusion; n = 6]. A and C, control bars (Pre; means ± S.E.M.; n = 6) represent basal release of CGRP and substance P (SP) preceding ischemia/reperfusion. *, P < 0.05, **, P < 0.01, from Pre by one-way ANOVA followed by Dunnett's post-test analysis. B and D, hearts were perfused with both CGRP_8–37 (100 nM) and CP99994 (100 nM) for 10 min preceding ischemia/reperfusion (n = 6). The antagonists remained present throughout reperfusion. Control bars (means ± S.E.M.; n = 6) represent basal release of CGRP and substance P preceding ischemia/reperfusion. *, P < 0.05, **, P < 0.01, from control by one-way ANOVA followed by Dunnett's post-test analysis.

Fig. 6.

Combined CGRP- and NK₁-receptor blockade exerts cardioprotective, anti-RAS effects in ex vivo guinea pig hearts subjected to ischemia/reperfusion. Coronary overflow of renin (C) and NE (B) and duration of reperfusion arrhythmias (VT/VF) (A) in isolated guinea pig hearts subjected to ischemia/reperfusion (i.e., 20-min global ischemia + 30-min reperfusion; n = 6). Other hearts were subjected to ischemia/reperfusion preceded by 10-min perfusion with both CGRP and substance P (SP) NK₁- receptor antagonists (i.e., CGRP_8–37 and CP99994, each at 100 nM; n = 4). Both antagonists remained present throughout the period of reperfusion. Bars are means (± S.E.M.) of renin and NE overflows during the first 6 min of reperfusion and duration of VT/VF. * and **, P < 0.05 and P < 0.01, respectively, from ischemia/reperfusion control by ANOVA followed by Dunnett's multiple comparisons test.

Fig. 7.

Toxic aldehydes elicit the release of substance P (SP) and norepinephrine from cardiac synaptosomes and renin from HMC-1 cells. A, substance P released from cardiac synaptosomes incubated with acetaldehyde for 10 min. Points represent means ± S.E.M. (n = 4–7). Basal release of substance P was 16.36 pg/ml ± 8.92, n = 11). B, renin release from HMC-1 cells incubated with acetaldehyde for 10 min. Points represent mean percentage increase (n = 5–18) above basal renin activity (i.e., 130 pg/h/mg protein ± 17.5; n = 14). C, mean percentage increase in NE release above a basal level of 1.62 ± 0.043 pmol/g in cardiac synaptosomes incubated with acetaldehyde for 10 min (mean ± S.E.M.; n = 30). *, P < 0.05, **, P < 0.01, from control by one-way ANOVA followed by Dunnett's post-test analysis.

Fig. 8.

Proposed functional link between sensory C-fibers and cardiac mast cells and its role in renin release and local RAS activation. A, chemical stimulation of cardiac sensory C-fibers with capsaicin elicits the release of neuropeptides (CGRP and substance P), which activate their respective receptors on cardiac mast cells, sequentially promoting their degranulation, renin release, RAS activation, ANG II formation, stimulation of AT₁-receptors on sympathetic nerves, and thus, NE release. B, similarly to capsaicin, ROS and toxic aldehydes (RCHO) generated during ischemia/reperfusion act upon sensory C-fibers, eliciting the release of neuropeptides that in turn causes degranulation of juxtaposed mast cells, evoking the release of renin, RAS activation, ANG II formation, and NE release from sympathetic nerves. ANG II and NE contribute to the generation of sudden cardiac death-type arrhythmias. ROS and RCHO can further amplify the arrhythmogenic threat by directly eliciting renin and NE release from mast cells and sympathetic nerve terminals, respectively.